Cytidine is a structural subunit of ribonucleic acid that consists of cytosine and the sugar ribose. Cytidine triphosphate (CTP), an ester of cytidine and triphosphoric acid, is a substance used in the cells to introduce cytidylic acid units into ribonucleic acids. CTP also reacts with nitrogen-containing alcohols to form coenzymes that participate in the formation of phospholipids.
CTP is obtained from uridine triphosphate (UTP) during the pyrimidine biosynthesis. The conversion of UTP into CTP is catalyzed by an enzyme called CTP synthase as follow:ATP+UTP+glutamine→ADP+Pi+CTP+glutamate
CTP synthase, also known under the name of UTP-ammonium ligase, catalyzes the ATP-dependent conversion of UTP in CTP using glutamine or ammonia as a nitrogen source (Long & Arthur. 1967; Kursula et al, 2006). CTP synthase is a polypeptide chain comprising two domains: the C-terminal involved in the hydrolysis of glutamine and in transferring the N-terminal NH3 group used for UTP amination and production of CTP (Weng et al, 1986; Kursula et al, 2006). GTP, in the presence of glutamine, is an allosteric activator of CTP synthase glutaminase activity (Levitzki et al. 1972).
CTP synthase, being a key enzyme involved in nucleic acid and phospholipids synthesis, is involved in cell growth, development, and in tumorigenesis. CTP synthase is also a key enzyme for immune system functionality, insufficiency loss of function of this enzyme leading to immune deficiency. CTP synthase activity inhibitors are already used as anti-cancer compounds but these inhibitors lack specificity. Specific inhibitors of CTP synthase activity might be used as immunosuppressive compounds, particularly to avoid graft rejection after graft transplantation.
In this respect, there is thus a need to detect and quantify CTP and CTP synthase activity.
Different methodologies have been proposed to quantify CTP synthase activity and most are based on the determination of the conversion of UTP into CTP, using either monitoring of changes in absorbance at 291 nm by spectrophotometry (Long et al., 1967), or directly quantifying the nucleotides after chromatographic separation (Van Kuilenburg et al, 1997) or after incorporation of radiolabeled UTP (Brockman et al., 1975). Radioisotope-based methods have the advantage of being very specific, but they are very long, costly and difficult to implement in hospital for routine use. Moreover, the required amount of cells for the implementation of these methods is high, about 106 cells (Cohen et Al.—2010). Methods for quantifying the glutaminase activity of CTP synthase have also been developed (Iyengar et al., 2002). These methods have a high sensitivity but do not provide information on CTP formation.
More recently, it was showed a relationship between deficiency of CTP synthase of type I and the occurrence of severe immune deficiency (Martin et al, 2014), in this study, Martin et al. used a method for quantifying intracellular nucleotides using reversed-phase chromatography coupled to tandem mass spectrometry. However, this method does not allow a good separation of CTP and UTP and is thus not appropriate for determining or quantifying CTP synthase activity. Since all the nucleotides triphosphates have a specific transition, they can be distinguished when using tandem mass spectrometry even when they are not chromatographically separated. However, the inventors have found that there is a 12% interference between UTP and CTP that could be detrimental for CTP dosage when there is an excess of UTP and especially for CTP synthase activity assays since the samples are incubated with a large excess of UTP.
Moreover, most prior art methods for detecting and quantifying CTP and CTP synthase activity are time consuming, i.e the duration of nucleotides separation (separation time) takes more than 15 minutes and the total duration of analysis (run time) takes often more than 40 minutes (Cohen et al, 2009, Ping et al., 2009, WO 2006/107775).
An additional problem of some of these methods (for example those described by Cohen et al., 2009) is that, during the cell extracts preparation, CTP synthase is denatured and it is thus not possible to further measure accurately its enzymatic activity.
More generally, it appears that current methods for detecting and quantifying CTP and CTP synthase activity are time consuming, costly, require samples with high cell numbers, conduct to proteins denaturation, and thus do not allow specific detecting or quantifying of CTP or CTP synthase activity.
Thus, there is a need to provide alternative methods for detecting or quantifying CTP or CTP synthase activity, which overcomes the drawbacks of prior art methods.
Particularly, it is necessary to provide a method allowing an optimal separation between CTP and other nucleotides in the sample (in particular UTP), having a high specificity, without proteins denaturation at the stage of cellular extracts preparation, being less time consuming and requiring lower cell numbers in cell sample compared to prior art methods, and having good intravariability and intervariability. It is also necessary that this method be appropriate to routine hospital implementation.